Project Summary (Abstract): The rise of vancomycin resistance is of particular importance since this antibiotic is one of few remaining options to treat enterococcal infections. Vancomycin functions by binding to the terminal D-Ala-D-Ala residues of the pentapeptide portion of the cell-wall precursor lipid II. By binding lipid II, vancomycin effectively sequesters the substrate required for synthesizing the protective peptidoglycan cell wall layer, leaving the bacteria susceptible to osmotic lysis. Vancomycin-resistant bacteria have acquired a set of genes for enzymes that reprogram lipid II biosynthesis to replace D-Ala-D-Ala with D-Ala-D-Lac, to which vancomycin cannot efficiently bind, diminishing vancomycin's ability to inhibit peptidoglycan synthesis. In resistant bacteria, the reprogramming enzymes are regulated by the VanR/VanS two-component system, and while the mechanism of resistance is understood, this regulation remains largely uncharacterized. The phosphorylated form of VanR is an activated transcription factor that initiates the transcription of the resistance genes to reprogram lipid II biosynthesis. VanS is a sensor histidine kinase that modulates VanR's phosphorylation state according to the presence of vancomycin. In the absence of vancomcyin, VanS acts as a phosphatase to maintain VanR in its inactivated state. In the presence of vancomcyin, VanS detects the antibiotic, and transduces a signal to keep VanR phosphorylated. It remains unclear how VanS detects vancomcyin, and how that detection alters VanS activity; the studies I propose aim to clarify these processes. I also plan to explore the molecular basis of different vancomycin-resistance phenotypes seen in different bacterial strains, for which the inducibility of the resistance genes varies widely. These studies will begin to provide a mechanistic understanding of how the extracellular vancomycin signal is transduced to generate an intracellular response. Finally, I also plan to address the largest knowledge gap in our understanding of two- component systems, which stems from the lack of structural information, by crystallizing portions of the VanR/VanS system. Studying the structure and function of the VanR/VanS system will help elucidate the mechanism of induction by vancomycin, likely identifying new therapeutic targets against resistant bacteria.